PROJECT SUMMARY This competing renewal of GM076710 involves research on bacterial cell wall biosynthesis, an essential and highly conserved pathway that is a well-validated target for antibiotics, including the beta lactams and the glycopeptides (e.g., vancomycin), two of the most important classes of clinically used antibiotics in history. Unfortunately, resistance to these and other clinically used classes of antibiotics has become a major problem and it is critical to develop new approaches to overcome resistant infections. Studies to understand known as well as new possible targets, in conjunction with methods to evaluate inhibition of these targets, may ultimately lead to new antibiotics. Hence, the overall goal of this renewal is to develop tools and methods to study the final steps of peptidoglycan assembly and apply them to characterize biosynthetic enzymes as well as cell wall inhibitors. These final steps of PG assembly are notoriously difficult to study because the chemical transformations involve large, complex molecules and some of the enzymes are integral membrane proteins. This renewal combines synthetic and enzymatic approaches to obtain substrates with enzymology, bacterial genetics, and cell biology to probe enzyme mechanism and inhibition both in vitro and in cells. There are four specific aims: 1) to develop tools to quantify pool levels of key intermediates in PG biosynthesis in order to verify inhibition of PG biosynthetic enzymes and determine mechanism of action of cell wall targeting antibiotics; 2) to develop substrates and assays to study bacterial transpeptidases and apply them to characterize PBP2a, the transpeptidase responsible for beta lactam resistance in Staphylococcus aureus, with the longterm aim of exploiting this knowledge to develop strategies to overcome PBP2a-mediated resistance; 3) to develop methods to study cell wall tailoring enzymes that attach teichoic acids and other glycopolymers to PG because these enzymes are possible targets for antibiotics; and 4) to develop methods to probe translocation of Lipid II and its inhibition in both Gram negative and Gram positive organisms because the Lipid II flippases are also possible targets for antibiotics.